A single subtumorigenic exposure to a carcinogen (initiation) and subsequent chronic regenerative epidermal hyperplasia of sufficient magnitude (promotion) can induce on the backs of mice benign and malignant cutaneous neoplasms. There are three striking characteristics of this process: i) initiation occurs quickly and is essentially permanent; ii) it is latent in the absence of promotion; iii) the tumor responses are similar regardless of the interval between initiation and promotion. The cells that maintain this "latent neoplastic lesion" have long been thought to be epidermal stem cells analogous to those of the bone marrow, but this has never been proved. The hypothesis of this proposed research is that the epidermis indeed has stem cells and that these are the progenitors of the cutaneous neoplasms of two-stage chemical carcinogenesis. The overall objective is to test this. In Aim 1, freshly isolated epidermal keratinocytes (including those from the hair follicles) from adult mice are sorted by flow cytometry following staining with a nontoxic vital dye such as rhodamine-123, and to determine whether they have characteristics of stem cells. Each sorted population is analyzed by light microscopic autoradiography for enrichment for label-retaining, as well as labelled keratinocytes. The sorted populations are also analyzed for their biological activity according to their ability to form colonies from single cells. Aim 2 is to determine by quantitative light microscopic autoradiography of culture dishes whether primary in vitro colonies with high reproductive capacity are founded from the label-retaining cells and not the pulse-labelled keratin-ocytes. Aim 3 is to determine whether the colonies with high reproductive capacity are serially subculturable and form a epithelium upon grafting to athymic animals. Aim 4 is to determine the fate of the stem cells and other proliferative keratinocytes during the production and regression of epidermal hyperplasia. Aim 5 is to determine whether the epidermal stem cells maintain the "latent neoplastic lesion" following in vivo exposure to a chemical carcinogen. An agent such as 5-fluorouracil is used in mice to kill cycling but not quiescent epidermal cells in the context of chemical carcinogenesis experiments and also in grafting studies with primary in vitro keratinocyte colonies. Achievement of Aims 1, 2, and 3 constitutes isolation of the epidermal stem cells from adult mice and proof of the epidermal stem cells hypothesis. Achievement of Aim 4 supports the proof by demonstrating that these cells behave in a biologically meaningful way during epidermal hyperplasia. Achievement of Aim 5 constitutes identification of the initiated cell following exposure to a carcinogen as an epidermal stem cell. This proposed investigation is significant for cutaneous pathobiology because the identification of the stem cells will lead to the identification of specific stromal cells and key factors that regulate normal and abnormal epidermal growth and differentiation and morphogenesis of the epidermal appendages in a way simply not possible with in vitro systems. This investigation is significant for cancer research in that it sets the stage for identification of the neoplastic lesions in specific target keratinocytes.